Retrievable bioartificial implants having dimensions allowing rapid diffusion of oxygen and rapid biological response to physiological change, processes for their manufacture, and methods for their use

ABSTRACT

Bioartificial implants and methods for their manufacture and use are described, particularly bioartificial pancreases. In particular, the implants may be thin sheets which enclose cells, may be completely biocompatible over extended periods of time and may not induce fibrosis. The high-density-cell-containing thin sheets are preferably completely retrievable, and have dimensions allowing maintenance of optimal tissue viability through rapid diffusion of nutrients and oxygen and also allowing rapid changes in the secretion rate of insulin and/or other bioactive agents in response to changing physiology. Implantations of living cells, tissue, drugs, medicines and/or enzymes, contained in the bioartificial implants may be made to treat and/or prevent disease.

“This application is a divisional of Ser. No. 09/128/188 Aug. 3, 1998U.S. Pat. No. 6,165,225, which is a divisional of Ser. No. 08/542,506,Oct. 13, 1995, U.S. Pat. No. 5,855,613.”

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to the field of bioartificialpancreases, bioartificial implants generally, and methods for theirmanufacture and use. In particular, the present invention is directed tothe fabrication of thin sheets which enclose cells, which are completelybiocompatible over extended periods of time and which do not inducefibrosis. The present invention also concerns cell-containing thinsheets which are easily completely retrievable, and which havedimensions allowing maintenance of optimal tissue viability throughrapid diffusion of nutrients and oxygen and also allowing rapid changesin the secretion rate of insulin and/or other bioactive agents inresponse to changing physiology. The present invention also concernsimplantations made using these bioartificial implants. The presentinvention may be used for implantation of living cells, tissue, drugs,medicines and/or enzymes, contained in the bioartificial implants.

2. Discussion of the Background

Traditional medical treatments for functional deficiencies of biologicalorgans have focused on replacing identified normal secreted products ofthe deficient organ with natural or synthetic pharmaceuticalcompositions. For example, for treating insulin-dependent diabetesmellitus (IDDM), also known as type I or juvenile diabetes, the normalsecretion of insulin by the islets of Langerhans of the pancreas must bereplaced since functional islets are no longer present in the pancreas.This islet function is emulated by administering insulin, titrating theinjections in response to blood glucose level measurements. The normalproduction of hormone by the islets is poorly approximated. This therapyis known to be associated with premature morbidity and mortality.

Organ replacement has also been applied. For IDDM, whole pancreas andislet transplants both have been performed with significant success¹.Such transplants require continuous use of immunosuppressive agents toprevent immunological rejection of the organ, depriving the patient ofthe full protective function of the immune system against disease andsubjecting the patient to the side effects of the drugs.

The bioartificial implant is the manufactured solution to replacingorgan function without reliance on immune suppression; this topic hasbeen reviewed^(2,3,4,5,6). Many approaches have been tried forfabrication of bioartificial implants having in common the use of somesemipermeable barrier allowing the tissue to function and simultaneouslyprotecting tissue from rejection. Prior to the present invention, suchbioartificial implants have proven unsatisfactory for a variety ofreasons.

Because of severe limits on availability of human tissue, a practicaltherapy for IDDM will likely require the use of xenografts, most likelyof porcine tissue. Porcine tissue is known to stimulate a vigorousimmune response in humans, including complement mediated rejection ofthe xenograft, posing a significant obstacle to use of porcine tissue ina bioartificial implant. The bioartificial implant barrier shouldsufficiently exclude antibody-complement to prevent complement mediateddamage to cell and tissue xenografts.

Some bioartificial implants require grafting into the vascular system,usually with connections to an artery and a vein to take advantage ofthe pressure difference. Blood flow through such vascular grafts hasproven to be traumatic to blood, and continuous systemic administrationof pharmaceutical preparations to prevent clotting and foreign bodyreactions is required. Thus optimal bioartificial implant design, notrequiring continuous systemic pharmaceutical administration, is limitedto implants that rely on passive diffusion.

Most such passive diffusion bioartificial implants fail because theirdimensions are such that the enclosed tissues cannot receive enoughnutrients, especially oxygen. When tissue is starved of oxygen itsmetabolism declines and it loses its ability to secrete hormone.Extended hypoxia leads to cell death.

In these same devices, the tissue may be prevented from responding in atimely way to changes in the physiological environment because of thedimensions of the bioartificial implant. In the case of implantscontaining insulin-producing islets, an unsatisfactory bioartificialimplant may not be able to sense that previously secreted insulin hassuccessfully reduced blood sugar levels, and thus, will continue tosecrete insulin even when the effect of this insulin is to plunge bloodsugar levels below normal, endangering the host.

Many bioartificial implants fail because their surface is notbiocompatible. When exposed to living tissues, especially when they havebeen seeded with allogeneic or xenogeneic living tissue, they provoke aforeign body response. The foreign body response may be caused by amaterial on the surface of the implant, antigens shed by the cellswithin, or by a combination of both. The foreign body response includesfibrosis, in which fibroblasts and macrophages apply proteins includingcollagen to the surface of the implant, attracting other effector cells,and eventually leading to the formation of a capsule of connectivetissue that isolates and starves the implant.

Some bioartificial implants are unsatisfactory because they cannot beeasily removed in the event of implant failure or dysfunction.

Some processes previously developed for fabrication of bioartificialimplants did not yield reproducible products having the desired porosityand thickness required for the implant.

Some processes previously developed did not completely cover the livingtissue, thus allowing access by host immune system to the living tissue,leading to foreign body reactions and/or sensitization and antibodyformation followed by complement-mediated lysis.

Some processes previously developed for bioartificial pancreas implantsdid not make efficient use of islets, wasting a significant fraction ofthem during fabrication.

Some bioartificial implants are so bulky that implantation is difficultor impossible. This problem occurs when only a small fraction of thevolume of the implant consists of living cells.

Prior to the present invention, all bioartificial implants have provenunsatisfactory for one or more of these reasons, and for other reasonsas well.

Ease of Complete Retrieval

The need for complete retrieval of the bioartificial implant in theevent of failure has been stressed by several workers in thefield^(7,8,9), especially Paul Lacy, a recognized leader in isletimplant research. He has criticized encapsulated islets⁹:

“To be feasible for broad use, the capsules would have to be smaller andmore stable. In addition, investigators would have to develop a way toretrieve all the capsules readily in the event removal becamenecessary.”

Another reason that the optimal bioartificial implant must be easilycompletely retrievable is that such devices will require review by theFDA's division of biologics before sales in the United States¹⁰. Suchreview examines all safety issues and risks, including ease of retrievalin the event removal becomes necessary. A clinical trial of abioartificial implant in the United States was done under an FDAapproved protocol¹¹.

Diffusion and Bioartificial Implant Dimensions

It is now widely recognized that a successful bioartificial implant musthave dimensions that permit efficient diffusion of nutrients into theimplant and secretion of bioactive agents out of the implant. Yet thevast majority of bioartificial implants described in the literature havedimensions which make efficient diffusion impossible, either becausethey were manufactured before the magnitude of the requirement wasunderstood, or because there was no art to manufature the implant withthe required dimentions.

Theoretical studies have predicted the maximum dimensions permissiblebased on the physics of diffusion. The maximum dimension is a functionof the density of tissue in the implant. Experimental studies confirmthe theoretical limits to passive diffusion and point to the parametersof implant dimension.

Diffusion both into and out of the bioartificial implant must beconsidered. Secreted products must diffuse out of the bioartificialimplant efficiently. Nutrients must diffuse in at a rate sufficient tosupport normal cellular metabolism. The rate-limiting nutrient isoxygen, and implants that die from lack of nutrients usually die ofhypoxia.

First, in an islet bioartificial implant, insulin must diffuse quicklyout of the device in response to changes in ambient glucose levels.Implants that are too large delay insulin response. For instance, afterglucose stimulus is removed, hollow fiber implants (730 μm O.D.)continue to secrete insulin long after glucose has normalized¹². Thisexcess insulin can result in potentially life-threatening low bloodsugar.

Experimental studies have shown that bioartificial implants that arelarger than 350 μm in their shortest dimension significantly delay therelease of insulin in response to glucose challenge^(13,14). Althoughstudies of insulin release kinetics in vitro alone are useful, glucosetolerance and insulin levels should be measured in vivo in an animalmodel to more conclusively determine the potential effectiveness of aparticular implant structure and design.

Second, the most difficult problem associated with implants relying onpassive diffusion is oxygen diffusion. The importance of oxygendiffusion has only been recognized relatively recently. Currenttheoretical models predict that oxygen diffuses to a depth of less than200 μm through living tissue^(5,15). The most detailed analysis of therelationship between implant dimensions, implant shape, engraftment,oxygen tension and islet viability has been published recently byColton⁵:

Maintenance of maximum cell viability and function is essential and, inthe absence of immune rejection, is limited by the supply of nutrientsand oxygen, especially the latter . . . The oxygen levels to which theislet cells are exposed are important from two standpoints, viabilityand function . . . Planar diffusion chambers, preferably with hightissue density, represent an attractive configuration forimmunoisolation devices. Diffusion chambers prepared with parallel, flatsheet microporous membranes were employed extensively several decadesago in early studies of immune rejection during transplantation.Invariably, the membranes employed produced an extensive fibroticforeign body response composed of an avascular layer of fibroblasts in acollagen matrix which restricted the supply of oxygen and nutrients,resulting in minimal survival of the encapsulated tissue . . . Only whenthe oxygen source is brought to within about 25 μm from the surface ofthe transplanted tissue does the pO₂ at the center of a 100 μm-thickslab stay above zero.

This analysis shows that diffusion of oxygen is crucial for isletviability and function and that the shortest dimension of the coating,capsule or slab/sheet permitting oxygen diffusion throughout thestructure ranges up from 100 μm depending on tissue density, implantconfiguration and the proximity of oxygenated tissue.

Studies of islets in hollow fibers, microcapsules and coatings havesupported these theoretical conclusions^(14,16,17,18). NMR/EPR studiesof O₂ concentration suggest that oxygen is low in alginate capsules¹⁹.These observations confirm that the configuration of the device must bevery thin. As a rule of thumb, each cell must be less than 200 μm fromthe surface of the bioartificial implant to receive oxygen, assumingthat the implant is in contact with well oxygenated tissue.

Biocompatible Materials

There is considerable confusion about the term “biocompatibility.” Thesciences concerned with implantable medical devices use the term verydifferently from the sciences concerned with implants. For a device,biocompatibility means that the material induces engraftment, e.g., thevascular graft is covered with a layer of collagen fibers. This permitsovergrowth of endothelial cells, which is desirable for a vasculargraft. In contrast, such overgrowth will starve a cellular implant.Thus, biocompatibility for a living implant means the material will notpromote cellular adhesion. The term “biocompatible” is used here to meanthat the material by itself or in combination with living tissues doesnot produce foreign body reaction or fibrosis.

Surprisingly few materials meet this definition. It is generallybelieved that, the foreign body reaction is initiated when proteinsadhere to the foreign body²⁰. Thus the material, to prevent the initialstep of protein adsorption, should be very hydrophilic. For example,almost every biocompatible polymer is a polysaccharide, a polyalcohol,an organic acid, or a combination thereof.

Most published work makes use of alginate, a polymer of alginic acids,which are sugar acids. Other biomaterials that are claimed to bebiocompatible include agarose²¹, agarose/poly(styrene sulfonic acid)²²,hydroxyethyl methacrylate-methyl methacrylate copolymer²³, polyvinylalcohol^(24,25), protamine-heparin²⁶ and others. One group hasphotopolymerized a skin of polyethylene glycol onto alginate to improveits biocompatibility²⁰; another used free radical polymerization ofethylene oxide and alginate to improve stability²⁷.

The most commonly used material for fabrication of bioartificialimplants has been alginate, usually in combination with a polycationsuch as poly-L-lysine to form a membrane. The originalalginate/polylysine capsule was shown to ameliorate diabetes in miceover fifteen years ago²⁸.

This occurrence of alginate in nature is mainly in the marine brownalgae (Phaeophyta), soil bacteria and Pseudomonas. Alginate is a familyof unbranched co-polymers of (1-4)-linked β-D-mannuronic acid (M) andα-L-guluronic acid (G). It is located in the intercellular algae matrixas a gel containing cations (predominantly Ca²⁺) that crosslink thepolymer fibers. Alginates appear to be block co-polymers comprised ofhomopolymeric blocks of M and G with regions of alternating MG. Methodshave been developed for purification of high M and high G fractions ofvarious molecular weights^(29,30,31,32) sufficiently pure to make thembiocompatible^(33,34,35,36).

Alginates obtained from marine algae comprise approximately 40% of thedry weight of the kelp. Because of its ability to retain water and itsgelling, viscosifying and stabilizing properties, alginate is widelyused industrially.

Until recently, alginate/polylysine capsules induced fibrosis and werenot biocompatible³. Grafts failed over periods of days and months. Suchgrafts were found to be covered with dense fibrous overgrowth.

These capsules caused fibrosis for several possible reasons. First,polylysine is fibrogenic. Capsules made with alginate alone are lessfibrogenic³⁷. In addition, researchers at the University of Gieβen andthe University of Würzburg^(36,35), and separately at the University ofCalifornia^(33,34,29), succeeded in showing that alginate fibrosis wascaused by impurities in alginate. Sufficiently purified alginate doesnot induce fibrosis. (The concept proposed by University of Trondheimthat high-guluronate alginates are biocompatible while high-mannuronateare not³⁸ has been shown to be incorrect^(37,39).) Thus, alginate can bemade biocompatible (as defined above) with a series of rigorouspurification steps.

One of the present inventors (Dorian) in collaboration with researchersat the University of California established that removal of sufficientcontaminants from alginates results in alginate fractions that arenonfibrogenic, regardless of the G, M and GM content^(33,34). It wasalso shown that the molecular weight and block contents can bemanipulated to produce alginates with desirable physical properties.Thus, it is now possible to produce both high M and high G alginatesthat are biocompatible with and without cells or islets.

Finally, it is worth noting that some workers in the field haveconcluded that biocompatibility, as we have defined it, is not necessaryfor a successful implant. Instead of a material that is ignored by thebody's foreign body reaction cells, they seek one that produces acertain type of foreign body reaction²:

Biocompatibility is most often achieved by a careful manipulation ofcomposition, purity, geometry, handling, surface morphology, andsurgical techniques to yield and implant and implant site which issufficiently inoffensive to the host to remain below the threshold ofstimulation required to provoke macrophage activation and fibroblastdeposition . . . Less frequently but also successfully, biocompatibilitycan be obtained by texturing the surface of the implant to encourage thegrowth of a host vascular network at the implant surface . . . Thisstrategy no longer requires membrane and materials which to not engendera foreign body response and consequently permits somewhat greaterlatitude in setting membrane transport requirements . . . This approachhas been tried in the past without success, and was patented⁴⁰.

We reject this approach because we believe that strict neutrality isdesirable. For us, “biocompatible” means that the material by itself orin combination with living tissues does not produce foreign bodyreaction or fibrosis.

Auto Inhibition of Product Secretion

Pancreatic islet cells in vivo receive arterial blood, and therefore,sense the same levels of nutrients and hormones in the blood prevalentelsewhere in the body. Islet cells in a bioartificial implant mayexperience higher levels of their own secreted hormones because they aredependent on diffusion to is disperse hormones. A bioartificial implantwhich inhibits or impedes diffusion of hormones will cause islet cellsto respond to an artificially high concentration of hormones. Forexample, secretion of insulin by islets is inhibited by high ambientlevels of insulin⁴¹. It may be advantageous to make the implant as thinas possible and thus aid the diffusion process.

Complement-Antibody Lysis of Implants

As described previously, passive diffusion of nutrients, oxygen andsecretory products such as insulin (M.W.=7 kD) should be uninhibited tomaximize the effectiveness of a bioartificial implant. This suggeststhat the membrane or coat of the bioartificial implant should have largepores. Larger pores, however, would admit antibodies (M.W.=160 kD), andlarger, if sufficiently, complement (M.W. [Clq]=390 kD). If antibody andcomplement can penetrate the coat or membrane, the cells inside theimplant are at risk of lysis and death.

Microcapsules have been made that inhibit antibody and complementdiffusion sufficient to prevent islet lysis and permit normalization ofblood sugar in mice. This was shown even when immune sensitization wasdemonstrated using MLC cultures³⁶. However, similar results in highermammals have not been reported.

The complement system, which exists in its most active (“complete”) formonly in higher mammals, is triggered by antibody and can kill foreigntissue in minutes⁴². Normally, allografts unprotected by bioartificialbarriers are killed by cellular attack, not complement attack. However,xenografts often trigger a vigorous antibody and complement response. Insome donor-recipient combinations (e.g., pig-to-dog), preformedantibodies exist and the complement response occurs minutes afterimplantation, before sensitization can occur.

The vast majority of islet implant studies have been performed usinginbred mouse strains as recipients. These animals are deficient atmounting a complement mediated response, and such studies are thus notpredictive of, for example, a human response to xenografts⁴³.

Because of ethical concerns and a shortage of suitable human tissue, onepractical therapy for human diabetes is a xenograft bioartificialimplant. Recent studies, however, have demonstrated thatporcine-to-monkey xenografts can trigger a vigorous antibody andcomplement response, raising grave concerns for porcine-to-humanxenografts^(44.)

The optimal bioartificial implant should prevent diffusion of antibodyand complement sufficiently well to prevent antibody and complementlysis of the living cells within the bioartificial implant. Microporousmembranes by their nature do not have a sharp molecular weight cutoff.Rather, the membranes increasingly inhibit diffusion as molecular weightincreases, in a manner represented by a sigmoid curve⁵. Thus, freediffusion of insulin and sufficient inhibition of complement diffusionrequire a subtle and complex “tuning” of the membrane. Experimentaldemonstration of this phenomenon caused the Gieβen group to comment⁴⁵:

“Our results lead to the concept of persistent immune interaction after‘immunoisolated’ transplantation. In this case a bioartificial pancreascould not be described as an immunoisolated islet transplantation but asan ‘artificially immunoprivileged site of transplantation.’”

In conclusion, the concept of immunoisolation should be supplanted by arecognition that the ideal implant is a subtle balance of multipledemands, including the molecular weight diffusion function of themembrane or coat, in one strategy to fabricate a successfulbioartificial implant.

Inefficient, Incomplete and Inconsistent Fabrication Methods

Islets are precious. A loss of fifty percent of the islets duringfabrication of the bioartificial implant would mean only half as manypeople with IDDM could be implanted. Some technologies are known to havea poor yield. For instance, air knife coating has a yield of 70%⁴⁶, andbarium alginate density gradient coating has a yield of 40%⁴⁷. Mostpublished methods do not report the yield, or survival rate, of islets.

Another form of inefficiency is a device design that includes too much“dead” space (i.e., space unoccupied by tissue or cells). Hollow fiberand disc designs have been reported to be 97% “dead” space⁸ 96% “dead”space¹⁴, respectively. In these configurations, the ratio of the deadspace to the islet volume is ≧25. In the optimal bioartificial implant,this ratio should be much smaller. In the limiting case, the implantwould be pure tissue.

As a result of imperfect coating, it is possible that islets mayprotrude from finished capsules, leading to fibrosis⁴⁸. Anotherconsequence of imperfect coating can be sensitization followed byantibody production, leading to complement lysis.

Defects in the capsules have been reported to lead to fibroticovergrowth⁴⁹. Some methods do not appear to be reproducible⁵⁰.

The optimal bioartificial implant fabrication method should bereproducible and have a high yield. The optimal bioartificial implantshould have a small volume ratio and no cells protruding, and be free ofphysical imperfections that lead to fibrosis.

Cellular Trophic Factors

The optimal bioartificial implant favors the health of the implantedcells. For instance, good glycemia helps all islet implants. It has beendemonstrated that islets retain more of their original mass followingimplantation when the glycemic level of the host is maintained nearnormal as the islets are engrafting⁵¹. It is therefore not surprisingthat clinical researchers report greater success when efforts were takento stabilize islet implant recipients' blood sugar levels⁵².

A number of reports suggest methods to improve islet cell mass,viability and functionality by including substances in the bioartificialimplant composition. Given the importance of oxygen in maintaining cellviability, it is not surprising that there is a report that hemoglobinimproves oxygen transport and thus islet viability¹⁸. Collagen also hasa reported benefit to islet viability, presumably because islets areattachment-dependent cells⁷. Other components of the extracellularmatrix including fibronectin, laminin, and tenascin are also recognizedas playing a role in cell growth and differentiation⁵³, and thus may besustaining.

Numerous trophic hormones and factors have appeared in the literature,including such trophic factors as insulin-like growth factor-I,insulin-like growth factor-II, transforming growth factor-alpha,hepatocyte growth factor/scatter factor, platelet-derived growth factor,and ilotropin, a paracrine/autocrine factor associated with isletdifferentiation.^(54,52) There is a report that antioxidant vitaminsreduce fibrotic overgrowth⁵⁵, and another that the vitamin nicotinamidehelps islet viability⁵⁶.

Another approach to maintaining cell viability and functionality is toco-culture with cells that secrete trophic factors⁵⁷.

The optimal bioartificial implant may include all such substances to theextent that they are compatible with other components and are beneficialto the implanted cells.

Useful Cells for Incorporation in Bioartificial Implants

Although the discussion herein focuses on islets of Langerhans fortreatment of diabetes, many uses of cellular implants to treat diseaseshave been proposed, including treatment of hemophilia, dwarfism, anemia,kidney failure, anemia, chronic pain, Alzheimer's disease, fulminanthepatic failure, Parkinson's disease, etc. This topic has beenreviewed.⁵⁸.

Previous Approaches

Numerous bioartificial implants have been described. The followingdiscussion is limited to passive diffusion type implants each containingmultiple islets, and does not include individually encapsulated isletsor vascular devices. (Two examples or capsules are included in the tablefor purposes of comparison.) In many cases dimensions and tissue densityare not reported in the reference, and we have noted the assumptionsused to generate numbers in the summary table at the end of the section.Oxygen at the center of the implant has been measured very rarely;oxygen levels were determined by applying the analysis of Colton at al.⁵

General reviews of immunoisolation technologies were noted earlier;discussions of planar congifurations—slabs, discs and sheets—have alsobeen published. For the most comprehensive and detailed on olderresearch, see Scharp⁵⁹, and for the most recent work, see the tworeviews by Colton^(4,5.)

Aebischer et al.⁶⁰ have described a tubular polymer implant made by“coextruding an aqueous cell suspension and a polymeric solution througha common port to form a tubular extrudate having a polymeric outercoating which encapsulates the cell suspension.” In one embodiment, thetubular extrudate is sealed at intervals to define separate compartmentsconnected by polymeric links. This system has a low cell density,reported at 0.1% in the patent example. The dimensions of the cylinderdevice do not permit oxygen to reach the center.

Andersson et al.⁶¹ describe a laminated double membrane disc designed tosimultaneously prevent cellular invasion and promote vascularization.The paper describes fibroblast invasion suggesting a lack ofbiocompatibility. While tissue density is high and the thickness issufficiently small to permit oxygen throughout the device, the smalldiameter of the disc results in insufficient volume per transplant. Thedesign does not appear to permit sufficient control over thickness ifthe diameter is larger.

Ash et al.⁶² have described a system comprising a bundle of fibers thatremains under the skin allowing replacement of cells. The dimensions ofthe sack-like device do not permit oxygen to reach the center if celldensity is greater than 10%.

Aung et al.²⁴ have described a biohybrid artificial pancreas utilizingmesh-reinforced polyvinyl alcohol hydrogel tubes as membranes. The tubeshave a 2 mm ID, and the membrane is 200 μm thick. The configurationprevents suitable kinetics and oxygen tension. As Aung's groupreported³³,

“. . . islets tend to aggregate into large clumps, and subsequentcentral necrosis occurs, probably due to the hypoxia of the centralportion, resulting in impairment or loss of function of the isletswithin 1 to 2 weeks.”

Thus, experimental results confirm that the thickness of the tubeproduces anoxia at the center.

Bae et al.⁶⁴ describe an implantable and refillable biohybrid artificialpancreas. The configuration is a disc shaped pouch. The polymer (acellulose acetate coated membrane) is not biocompatible. The dimensionsof the disc device do not permit oxygen to reach the center if celldensity is greater than 10%.

In a study that measured oxygen tensions in an acrylic tube implant,Bodziony¹⁶ found that the amount of oxygen present is too low to supportviability of islets. The amount of oxygen present was calculated to bedepleted by islet metabolism in less than three minutes.

Gaskill⁶⁵ proposed a passive diffusion “intravascular artificial organcomprised of a flexible, hollow, semipermeable catheter containingliving cells or tissue.” It is now known that the proposed materialsinduce thrombosis when placed in flowing blood. Although dimensions arenot disclosed, a tube with sufficiently small diameter to permit oxygendiffusion to the center would be several meters long, much longer thanone shown in FIG. 1 of the patent. Thus the invention of Gaskill doesnot have dimentions allowing oxygen to reach the center if cell densityis greater than 10%.

Inoue et al.²⁵ constructed a tube of polyvinyl alcohol membrane. Thedimensions of the cylinder device do not permit oxygen to reach thecenter if cell density is greater than 10%.

Jordan⁶⁶ describes a sack/tube, and even says that “the length of theshortest diffusion path between the interior side of the container walland the center of the glandular tissue must not be longer on the averagethan two millimeters.” However this length is not correct based oncurrent understanding of oxygen diffusion through tissue, and it is alsoincorrect to neglect the effects of diffusion through the containerwall. The Jordan design would have no oxygen in its center if celldensity is greater than 10% due to its dimensions.

⁶⁶Jordan, G. P. W., Artificial gland. U.S. Pat. No. 3,093,831 (1963)

Kopchick et al.⁶⁷ use hollow fibers seeded with genetically engineeredcells to deliver bovine growth hormone. The dimensions of the cylinderdevice do not permit oxygen to reach the center if cell density isgreater than 10%.

⁶⁷Kopchick, J. J., Leung, F. C., Livelli, T. J., and Malavarca, R. H.,Encapsulated mouse cells transformed with avian retrovirus-bovine growthhormone DNA, and a method of administering bgh in vivo; administer byhollow fiber filled with recombinant mouse cell suspension; lactation.U.S. Pat. No. 4,686,098 (1987).

Lacy et al.⁸ studied hollow fibers fabricated from an acrylic copolymerencapsulating small numbers of rat islets immobilized in an alginatehydrogel. The dimensions of the cylinder device do not permit oxygen toreach the center if cell density is greater than 10%.

⁸Lacy, P. E., Hegre, O. D., Gerasimidi-Vazeou, A., Gentile, F. T., andDionne, K. E., Maintenance of Normoglycemia in Diabetic Mice bySubcutaneous Xenografts of Encapsulated Islets. Science, 1991. 254: p.1782-1784.

Lanza et al.⁶⁸ used Amicon XM-50 membrane tubes sealed at the ends. Thedimensions of the cylinder device do not permit oxygen to reach thecenter if cell density is greater than 10%.

⁶⁸Lanza, R. P., Borland, K. M., Lodge, P., Carretta, M., Sullivan, S.J., Muller, T. E., Solomon, B. A., Maki, T., Monaco, A. P., and Chick,W. L., Treatment of severely diabetic pancreatectomized dogs using adiffusion-based hybrid pancreas. Diabetes, 1992. 41(7): p. 886-9.

Lanza, R. P., Sullivan, S. J., and Chick, W. L., Perspectives inDiabetes: Islet transplantation with immunoisolation. Diabetes, 1992.41: p. 1503-10.

Laue et al.⁵⁴ studied a regenerated cellulose hollow fiber system. Thedimensions of the cylinder device do not permit oxygen to reach thecenter if cell density is greater than 10%.

⁵⁴Miettinen, P. J., Otonkoski, T., and Voutilainen, R., Insulin-likegrowth factor-II and transforming growth factor-alpha in developinghuman fetal pancreatic islets. J Endocrinol, 1993. 138(1): p. 127-36.

Loeb⁶⁹ describes an “artificial endocrine gland for supplying a hormoneto a patient including an implantable housing placed in the body andhaving an impermeable extracorpeal segment and a semipermeablesubcutaneous segment [with a] replaceable envelope containing livehormone-producing cells such as pancreatic islet cells.” Althoughdimensions are not disclosed, a tube with sufficiently small diameter topermit oxygen diffusion to the center would be many meters long, muchlonger than one shown in FIG. 8 of the patent. Thus the invention ofLoeb does not have dimentions allowing oxygen to reach the center ifcell density is greater than 10%.

⁶⁹Loeb, M. P., Artificial endocrine gland containing hormone-producingcells. U.S. Pat. No. 4,378,016 (1983).

Ohgawara et al.⁵⁵ describe a diffusion chamber, but dimensions are notgiven. A cell density of 2% is described.

⁵⁵Laue, C., Zimmermann, U., Biesalski, H. K., Beyer, J., andSchrezenmeir, J., Antioxidative vitamin reduce fibrous tissue overgrowthof the bioartificial pancreas. Transplant Proc, 1995 27(2): p. 1875-6.

Penfornis et al.⁷⁰ also made use of the Amicon XM50 hollow fiberdescribed by Lanza. The dimensions of the cylinder device do.not permitoxygen to reach the center if cell density is greater than 10%.

⁷⁰Penfornis, F., Icard, P., Gotheil, C., Boillot, J., Cornec, C.,Barrat, F., Altman, J. J., and Cochin, J. V., Bioartificial pancreas inpigs. Horm Metab Res Suppl, 1990. 25(2): p. 200-2.

Scharp et al.¹¹ studied hollow fibers fabricated from an acryliccopolymer. Human islets were macroencapsulated and allotransplantedsubcutaneously. Recipients were patients with type I or type II diabetesand normal control subjects; none was immunosuppressed. The dimensionsof the cylinder device do not permit oxygen to reach the center if celldensity is greater than 10%.

¹¹Scharp, D. W., Swanson, C. J., Olack, B. J., Latta, P. P., Hegre, O.D., Doherty, E. J., Gentile, F. T., Flavin, K. S., Ansara, M. F., andLacy, P. E., Protection of encapsulated human islets implanted withoutimmunosuppression in patients with type I or type II diabetes and innondiabetic control subjects. Diabetes, 1994. 43(9): p. 1167-70.Performed under investigator FDA IND (BB-IND 5103).

Scharp has also developed planar diffusion chambers which have beendescribed by others (see table).

Schrezenmeir et al.¹⁸ used regenerated cellulose capillary membranes.The principal object of the study was to show that hemoglobin helpedmaintain viability by conducting oxygen. The dimensions of the cylinderdevice do not permit oxygen to reach the center if cell density isgreater than 10% in spite of the hemoglobin.

¹⁸Schrezenmeir, J., Kirchgessner, J., Gero, L., Kunz, L. A., Beyer, J.,and Mueller-Klieser, W., Effect of microencapsulation on oxygendistribution in islets organs. Transplantation, 1994. 57(9): p. 1308-14.

Tze et al.⁷¹ investigated polysulfone fibers in a coil configuration,and found massive fibrosis in only a few days. The dimensions of thedevice do not permit oxygen to reach the center if cell density isgreater than 10% center.

⁷¹Tze, W. J., Cheung, S. S., Tai, J., Bissada, N., Tsang, A., and Yep,W., Prolongation of pig islet xenograft survival in polysulfone fibercoil. Transplant Proc, 1994. 26(6): p. 3510-1.

Yang et al.¹⁴ used agarose to fabricate transplants in microbead, rodand slab configurations.

¹⁴Yang, H., Iwata, H., Shimizu, H., Takagi, T., Tsuji, T., and Ito, F.,Comparative studies of in vitro and in vivo function of three differentshaped bioartificial pancreases made of agarose hydrogel. Biomaterials,1994. 15(2): p. 113-20.

Various kinds of bioartificial pancreas (BAP) have been developed in thepast. There have been many disputes about the advantages anddisadvantages of each BAP. However, little attention has been paid tothe shape of the devices. In this study, three different shaped BAPswere made of the same material, agarose hydrogel. These are microbead-,rod-and disc-shaped BAPs, which are comparable to microcapsules, hollowfibre diffusion chambers and disc-shaped diffusion chambers,respectively, in shape. Numerical analyses showed that insulin releasekinetics depended greatly on the thickness of the gel membrane but noton the shape of the agarose hydrogel. These results suggest that theshape of the BAPs determines the in vivo functioning period of the BAPsand that the microbead is the most suitable shape for the BAP. Theauthors reported that “small differences such as oxygen tension andnutrients supply drastically affect the fate of encapsulated islets.”Theoretical modeling shows that the dimensions of all three implants donot permit oxygen to reach the center if cell density is greater than10%.

The following table summarizes prior approaches, giving severalquantities for comparison:

total volume per implant is given in cubic millimeters (μl); the volumeis determined for the entire transplant including membranes as well ascore tissue density is given as a percent of the total volume;

assuming an islet diameter of 150 μm when islet number is given orassuming 2×10⁸ cells per μl when cell number is given oxygen isdetermined at the center of the implant furthest from the surface; usingmeasurements (when available) or the equations of Colton et al.⁵assuming 10% cell density retrievability, whether the implant(s) caneasily be removed

⁵Colton, C. K., Implantable Biohybrid Artificial Organs. CellTransplant, 1995. 4(4): p. 415-436.

critical other islets per tissue O2 at dimention dimention volumetransplant density center Retrievable shape (mm) (mm) (mm3) count (%)Y/N Y/N spheres coatings and capsules diameter (mm) Dorian³³ coating0.32    0.02    1 10.3%  Y N Sun⁷² capsule 0.7     0.18    1 1.0% N Ncylinders tubes and fibers diameter length (mm) (mm) Aebischer⁵⁹ tube0.5   8  2 NA 0.1% Y Y Ash⁶¹ tube 50    75 147188   NA NA N Y Aung²⁴tube 2.4  40 181   1000 0.8% N Y Bodziony¹⁶ tube 1   20 16   200 1.8% NY Gaskill⁶⁴ tube NA NA NA NA NA N Y Inoue²⁵ tube 2.5  40 196   2000 1.4%N Y Jordan⁶⁵ tube 4   NA NA NA NA N Y Kopchick⁶⁶ tube 1.2  10 11 NA 0.1%N Y Lacy⁸ tube 0.73 20  8  1000 16.9%  N Y Lanza⁶⁷ tube 1.22 20 23   3161.9% N Y Laue⁵⁴ tube 0.62 30  9    5 0.1% N Y Loeb⁶⁸ tube NA NA NA NA NAN Y Penfornis⁶⁹ tube 1.22 20 23   316 1.9% N Y Scharp¹¹ tube 1   15 12  50 0.6% N Y Schrezenmeir¹⁸ tube 0.62 30  9   10 0.2% N Y Tze⁷⁰ tube1.1  25 24   25 0.1% N Y Yang¹⁴ tube 0.9  20 13   100 1.1% N Y planarconfigurations sheets, discs and slabs thickness diameter (mm) (mm)Andersson⁶⁰ disc 0.28  6  8   400 7.1% Y Y Bae⁶³ disc 1   40 1256  20000 2.2% N Y Ohgawara⁵⁵ disc NA NA NA NA 2.0% N Y Scharp* disc 0.22 6  6  2000 45.4%  Y Y Scharp* disc 0.22  6  6   500 11.3%  Y Y Yang¹⁴disc 2   15 353    600 0.2% N Y THIS INVENTION sheet 0.3  50 589  14614435.0%  Y Y This work is unpublished under David Sharp's name; however,some data and micrographs have appeared in a popular account of islettransplant research (note 9) and as “some very recent preliminaryresults” cited in Colton's review (5). The disc with 2000 isletscorresponds to FIGS. 16a and b in Colton, and the disc with 500 isletscorresponds to FIG. 16c, in which each islet is individuallyencapsulated before being put in the disc, accounting for the lowertissue density.

Prior to the present invention, no bioartificial implant was both (1)small enough in linear dimention to allow effective passive oxygendiffusion into medium density tissue and (2) large enough in volume tocontain over 200 islets.

NOTES

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Federlin, K. F., Islet transplantation. The connection of experiment andclinic exemplified by the transplantation of islets of Langerhans, Exp.Clin Endocrinol, 1993. 101(6): p. 334-45.

2. Lysaght, M. J., Frydel, B., Gentile, F., Emerich, D., and Winn, S.,Recent progress in immunoisolated cell therapy. J Cellular Biochem,1994. 56: p. 196-203.

3. Clayton, H. A., James, R. F., and London, N. J., Isletmicroencapsulation: a review. Acta Diabetol, 1993. 30(4): p. 181-9 .

4. Colton, C. K. and Avgoustiniatos, E. S., Bioengineering indevelopment of the hybrid artificial pancreas. J Biomech Eng, 1991.113(2): p. 152-70.

5. Colton, C. K., Implantable Biohybrid Artificial Organs. CellTransplant, 1995. 4(4): p. 415-436.

6. de Vos, P., Wolters, G., Fritschy, W., and Van Schiltgarde, R.,Obstacles in the application of microencapsulation in islettransplantation. Int J Artif Organs, 1993. 16: p. 205-212.

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Reach, G., Bioartificial pancreas. Transplant Proc, 1994. 26(2): p.397-8.

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Chen, C. F., Chern, H. T., Leu, F. J., Chang, T. M., Shian, L. R., andSun, A. M., Xenotransplantation of microencapsulated canine islets intodiabetic rats. Artif Organs, 1994. 18(3): p. 193-7.

SUMMARY OF THE INVENTION

The present invention concerns a bioartificial implant and methods forits manufacture and use. The dimensions of the bioartificial implant aresuch that cell viability may be maintained by passive diffusion ofnutrients, and preferably, such that a high cell density can bemaintained. The dimensions of the bioartificial implant are also suchthat the bioartificial implant is macroscopic and is easily retrievablefrom the host and is large enough to contain a significant fraction ofthe tissue required to achieve the desired therapeutic effect. Thepermeability of the bioartificial implant is such that cell viabilitymay be maintained by passive diffusion of nutrients, and preferably suchthat passive diffusion of secreted cell products permits rapid responseto changing physiological conditions. At the same time, the permeabilityof the membrane sufficiently impedes diffusion of antibody andcomplement to prevent lysis of the implanted cells, even when the tissueis a xenograft. The bioartificial implant is biocompatible, meaning itproduces no foreign body reaction.

The dimensions of the present bioartificial implant, when in a thinsheet configuration, are such that the surface area of a side of a sheetis at least 30 mm², preferably at least 2.5 cm² and more preferably 10cm², as defined by either (a) the diameter (if the sheet is circular) or(b) the area determined by the method of converging polygons. Althoughthe maximum dimensions can be that which is tolerated by the patientinto whom the implant is placed, for ease of fabrication and economy ofimplanted cells, the maximum surface area of a side of the present thinsheet implant may be 400 cm², more preferably 300 cm² and mostpreferably 250 cm² (for a human patient).

In the present bioartificial implant, the cell density is that which canbe contained within a thin alginate gel coat. Preferably, the celldensity is at least 10%, more preferably 20% and most preferably atleast 35% by volume.

The surface of the bioartificial implant is biocompatible. We have foundthat attempts to induce neovascularization at the surface of the implantwith synthetic polymers^(60,3) are less than ideal for long term implantviability. We have found that implants that are neutral (causing neitherfibrosis nor neovascularization) have been shown to last over a yearwith minimal decay of function¹².

⁶⁰. Aebischer, P. and Wahlberg, L., Method of encapsulating cells in atubular extrudate; coextrusion of semipermeable polymeric membrane andcells. U.S. Pat. No. 5,284,761 (1994). For the comparison table, weestimated the tube diameter and length from the figures and discussion(column 6, lines 15-17). Tissue density is based on 1×10⁵ cells/mlreported in the patent (column 8, line 10) compared with packed tissuedensity of 2 to 5 ×10⁸ cells/ml). ³. Clayton, H. A., James, R. F., andLondon, N. J., Islet microencapsulation: a review. Acta Diabetol, 1993.30(4): p. 181-9 . ¹². In reference 8, hypoglycemia during GTT wasdisclosed in the question period following presentation of this paper.

The bioartificial implant is described using the terms “cord,” “coat”and “overcoat.” The core comprises the living tissue, trophic factorsand nurse cells, alginate polymer crosslinked with a multivalent cationsuch as calcium, and a fiber mesh for strength. The coat comprisesalginate polymer crosslinked with a multivalent cation that serves tocontrol permeability. The overcoat comprises alginate polymercrosslinked with a multivalent cation that serves to render thebioartificial implant biocompatible.

One aspect of the present invention is its shape or configuration.Previous inventions have responded to inherent limitations on diffusionof oxygen by lowering cell densities within the bioartificial implant.We have found that an effective implant must have medium to high tissuedensities to minimize the volume of the total bioartificial implant, andthat the thickness of a sheet or slab must be very small to permiteffective oxygen diffusion. The sum of the core, coat and overcoatthicknesses should be less than 400 μm, preferably 350 μm or less, andmore preferably no more than 300 μm. The coat and overcoat thicknessshould be minimized so that the tissue quantity may be maximized. Wehave found that the coat and overcoat thicknesses may be from 10 to 100μm thick, preferably from 10-80 μm, and most preferably from 10-50 μm.Biocompatible implant coat thicknesses of only tens of μm have neverbeen before reported. The length and width of the bioartificial implanton the other hand should be maximized to permit the greatest possiblevolume of living tissue to be included in the bioartificial implant andto permit easy retrieval.

Another aspect of the present invention is the material out of which thebioartificial implant is constructed. Biocompatible purified alginateshave been described, but have only been used to produce coated andencapsulated implants. No bioartificial implant has been described usingalginate in a thin sheet. The properties of alginate gel can becontrolled by selecting the G and M content, the chain length, thealginate concentration, and counterions (e.g. calcium, zinc, barium or acombination thereof). Alginate in the core is compatible with livingtissue. The fabrication of alginate hydrogels is compatible with cellviability. Sodium alginate used to fabricate the core can be mixed withtropic factors and nurse cells along with the secreting tissue beforegelation with multivalent cations to provide a supportive environment.Alginate for the coating can be selected as to chain length and G and Mfractions and be gelled with various multivalent cations to control thepermeability of the coat. Alginate for the overcoat can be selected soas to maximize the biocompatibility of the bioartificial implant.

The bioartificial implant is fabricated using a series of moldsconstructed with frit materials that can be molded or milled, andmembranes. This allows diffusion of chelating agents (e.g., sodiumcitrate) or multivalent cation gelling agents (e.g., calcium, barium orzinc chlorides) to liquify and gel, respectively, the core, coat andovercoat. The manufacturing process makes use only of ions known to becompatible with the implanted cells.

Although many variations of the method exist, the essence of the processis simple. The shape of the core, coat and overcoat are molded while thealginate is liquid. The core and coat (and later the coat and overcoat)are crosslinked by simply contacting liquid alginate containing a smallamount of chelating agent to the gelled layer. The chelating agentdiffuses into the gelled layer and partially liquifies it. When acationic crosslinking agent is subsequently added, a tight bond isformed between the layers. The outer surface is made very smooth throughthe simple step of wetting the mold with crosslinking agent solutionbefore contacting the mold with the liquid coat or overcoat. We havefound that contact with the membrane thus wetted immediately produces avery smooth surface.

Because the coat and overcoat are fabricated from liquids, they can bemade very thin, even as thin as a few μm.

We have invented a bioartificial implant of dimensions never beforeachieved, with many attendant advantages. For example, one object of thepresent invention is to provide a bioartificial implant that is easilyretrievable from the host.

Another object of the present invention is to provide a bioartificialimplant in a thin sheet configuration permitting both high tissuedensities and diffusion to the tissue cells of the amounts of nutrients,oxygen and other substances required for cellular health, longevity andeffective function after implantation.

Another object of the present invention is to provide a bioartificialimplant in an easily retrievable thin sheet configuration, permittingboth high tissue densities and effective diffusion of nutrients into andcellular products out from the implant.

Another object of the present invention is to provide a bioartificialimplant in the configuration of a thin sheet of viable, physiologicallyactive, tissue cells for implantation which is physiologicallyacceptable to the host and which effectively provides prolongedprotection of the tissue cells, after implantation, from destruction bythe host immune system.

Another object of the present invention is to provide an effectiveimplant coating material in a retrievable thin sheet configuration whichis physiologically acceptable, non-fibrogenic and non-toxic to hosttissue.

Another object of the present invention is to provide a bioartificialimplant containing trophics such as nurse cells, ground substances,nutrients, hormones or oxygen carriers to support the cellular health,longevity and effective function of the implant after implantation.

A further object of the present invention is to provide a novelmanufacturing process for effectively enclosing an implant (e.g., cells,tissues and/or other biological substances) with a barrier or membranewhich is physiologically acceptable, non-fibrogenic and non-toxic tohost tissue, which may provide a complete barrier coating with acontrolled thickness and controlled permeability to intermediately sizedproteins, and which is easily retrievable.

In summary, the present invention may comprise an implant core having athin sheet configuration comprising viable, physiologically active,tissue cells and a crosslinked alginate gel and optionally, trophicfactors and nurse cells, and optionally, a fiber mesh support. Thealginates are preferably free from fibrogenic concentrations ofimpurities.

The bioartificial implant may have a coat and overcoat to controlpermeability and enhance biocompatibility. The implant sheet is thin andmay be permeable enough to provide a physiologically acceptable oxygentension at the center of the sheet when implanted in a suitable site ina human or animal subject. The thinness and permeability of the implantallows diffusion of nutrients, oxygen, metabolic waste products andsecreted tissue products. The implant preferably inhibits diffusion ofantibody and complement.

Preferably, the coat permits rapid diffusion of substances below 50 Kdand significantly inhibits diffusion of substances over 100 kD. The coatis also very thin, preferably approximately 10-50 μm. The coat maycomprise and thus, be constructed with, for instance, short chain, highM alginate gelled with a mixture of calcium and zinc ions.

Suitable cells for the present implant include, for example, pancreaticislets, renal cortex cells, parathyroid cells, thyroid cells, adrenalcells, hepatic cells, cells of various origins that have beengenetically engineered to secrete useful substances, cells of variousorigins that have been genetically engineered to metabolize harmfulsubstances, tissues, and the like.

The process of the present invention for making a biocompatible implantmay comprise the steps of:

(a) forming a core containing viable, physiologically active, tissuedonor cells in an alginate gel, preferably in a thin, sheet-shaped moldand more preferably by crosslinking an alginate solution containing thecells;

(b) contacting the core with a second alginate solution containing aconcentration of citrate sufficient to induce at least partialliquification of the alginate gel in the core;

(c) crosslinking the second alginate solution and liquified alginate gelin the core to form a coat layer bonded to the core, and thus, thebiocompatible implant, preferably by diffusing a crosslinking agent intothe second alginate solution and liquified alginate gel in the core.

Optionally, crosslinking step (c) may comprise contacting the innersurface of the mold with an aqueous calcium ion solution to create asmooth outer surface on the biocompatible implant. This optional stepmay be performed in addition to the diffusing step. A further option isto repeat step (b) with a third alginate solution containing sufficientcitrate to induce at least partial liquification of the alginate gel inthe coat, and repeat step (c) by crosslinking the third alginatesolution and liquified alginate gel in the coat to form an overcoatlayer bonded to the coat. The coat of the biocompatible implant may beformed to control permeability, and the overcoat formed to enhancebiocompatibility.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a schematic diagram of the thin sheet bioartificial implant.

FIG. 2 is an enlarged schematic diagram cross section of the thin sheetbioartificial implant shown in FIG. 1.

FIG. 3 is an enlarged schematic diagram cross section of the core edgeand the coating of the thin sheet bioartificial implant in the coatingmold.

FIG. 4 is an enlarged schematic diagram cross section of the core of thethin sheet bioartificial implant in the core mold.

FIG. 5 is an enlarged schematic diagram cross section of the coated thinsheet bioartificial implant in the coating mold.

FIG. 6 is an enlarged schematic diagram cross section of the overcoatedthin sheet bioartificial implant in the overcoating mold.

FIG. 7 is a schematic diagram cross section of the mold used forfabrication of the core, coating, and overcoating, shown in separatedsections (A), touching (B), and clamped (C).

FIG. 8 is a schematic diagram cross section of the mold used forfabrication of the coating half, shown in separated sections (A) andtouching (B).

FIG. 9 is a micrograph of the bioartificial implant core produced usingthe present invention using polymer spheres to represent islets (˜100X).

FIG. 10 is a micrograph of a section of the bioartificial implant coreproduced using the present invention using polymer spheres to representislets (˜100X).

FIG. 11 is a micrograph of a section of the complete (coated,overcoated) bioartificial implant produced using the present inventionusing polymer spheres to represent islets (˜100X).

DETAILED DESCRIPTION OF THE INVENTION

The bioartificial implant of the present invention is effective forimplantation into a host animal by standard surgical procedures or byuse of abdominal trochar and laparoscope.

The term “implant,” as used herein, is defined to include all livingtissues, cells, and biologically active substances intended to beimplanted into the body of a host animal, as well as the act ofimplanting or transferring these tissues and cells into a host. Thesetissues and cells include, without limitation, tissue and cells removedfrom a donor animal, tissue and cells obtained by incubation orcultivation of donor tissues and cells, cells obtained from viable celllines, cells obtained by genetic engineering, biologically activeproducts of cells and tissues, pharmaceuticals, drugs, enzymes,eutrophics, and the like. Tissues may perform a useful biologicalfunction either by secreting a therapeutic or trophic substance or byremoving a toxic or harmful one. An example of the latter would beremoval of various fatty substances from serum to reduce blood lipidlevels.

Any type of tissue or cells for which implantation is desired can befabricated into a sheet and implanted according to the presentinvention. The most common tissues for implants are secretory organtissues, where implantation from a donor organ to a recipient or hostanimal is desired to at least partially replicate the donor organ'saction in the host system. Preferred donor tissues are pancreaticislets, hepatic cells, neural cells, renal cortex cells, vascularendothelial cells, thyroid cells, parathyroid cells, adrenal cells,thymic cells and ovarian cells.

The process of the present invention is described hereinafter for thepreparation and implantation of pancreatic islets and islet cells by wayof example for purposes of clarity of explanation and not by way oflimitation. This process can be equally well applied to other organtissues as will be readily apparent to a person skilled in the art, withconventional and obvious modification as desired or required toaccommodate any uniquely different requirements of the differenttissues. Applications of the process to all tissues and cells suitablefor implantation are intended to be within the scope of the presentinvention.

Isolated pancreatic islets (or other cells or tissues suitable forimplantation) are prepared by conventional procedures to substantiallyseparate them from extraneous tissue and other donor substances.

In a first step of the process of the present invention, isolatedpancreatic islets (or other cells or tissue) are washed with isotonicsaline and suspended in solution of purified sodium alginate. Thealginate has been purified to make it fully biocompatible as describedin prior publications^(34,33,35).

³⁴Cochrum, K., Jemtrud, S., and Dorian, R., Successful Xenografts inMice with Microencapsulated Rat and Dog Islets. Transplant Proc, 1995.IN PRESS. ³³. Dorian, R., E. , Cochrum, K., C. , and Jemtrud Susan, A.,Non-fibrogenic high mannuronate alginate coated transplants, processesfor their manufacture, and methods for their use. U.S. Pat. No.5,429,821 (1995) ³⁵Zimmermann, U., Klock, G., Federlin, K., Hannig, K.,Kowalski, M., Bretzel, R. G., Horcher, A., Entenmann, H., Sieber, U.,and Zekorn, T., Production of mitogen-contamination free alginates withvariable ratios of mannuronic acid to guluronic acid by free flow,electrophoresis. Electrophoresis, 1992. 13(5): p. 269-74.

The alginate may be prepared with various molecular weights (chainlengths) high in guluronate residues or high in mannuronateresidues^(30,31,35). These methods are based on differential binding ofhomopolymeric M and G blocks to various cations. Selection of (i) thefraction of homomeric G blocks, homomeric M blocks and alternating GMblocks, (ii) the average chain length, (iii) the alginate concentrationand (iv) the mixture of cations used to gel the alginate can be used toproduce alginate gels with a very large variety of properties. In thefollowing, “alginate” means a solution of alginate selected for desiredproperties and which may be gellable, “alginate gel” means a crosslinkedalginate, and “multivalent cation” means a mixture of multivalentcations such as calcium, barium and zinc selected to give desiredproperties when used in combination with the selected alginate.

³⁰Martinsen, A., Storre, I., and Skjak-Braek, G., Alginate asimmobilization material: III. Diffusional properties. Biotechnology andBioengineering, 1992. 39: p. 186-194. ³¹. Skjak-Braek, G., Alginates:biosyntheses and some structure-function relationships relevant tobiomedical and biotechnological applications. Biochem Soc Trans, 1992.20(1): p. 27-33. ³⁵. Zimmermann, U., Klock, G., Federlin, K., Hannig,K., Kowalski, M., Bretzel, R. G., Horcher, A., Entenmann, H., Sieber,U., and Zekorn, T., Production of mitogen-contamination free alginateswith variable ratios of mannuronic acid to guluronic acid by free flow,electrophoresis. Electrophoresis, 1992. 13(5): p. 269-74.

The core alginate is selected for low viscosity in the liquid state (soas not to damage islets during core fabrication), high strength in thegel state (for a strong implant), compatibility with tissue, andcompatibility with trophic factors. The core alginate typically consistsof alginate (e.g., of from 80 to 800 kD, preferably from 100 to 500 kD,and more preferably from 200 to 400 kD) in a low concentration(preferably from 0.5 to 10%, more preferably from 1.0 to 5% and mostpreferably about 2.0%), having mixed M and G blocks crosslinked withcalcium.

The coat alginate is selected for high viscosity in the liquid state(particularly for the “gum” method), high strength in the gel state (fora strong implant), high permeability for low molecular weight species(e.g., permitting diffusion of molecules and/or complexes having amolecular weight below 150 kD, preferably below 100 kD, and morepreferably below 75 kD) and low permeability for high molecular weightspecies (e.g., inhibiting or prohibiting diffusion of molecules and/orcomplexes having a molecular weight above 200 kD, preferably above 150kD, and more preferably above 100 kD). Typically, the coat alginateconsists of short chain alginate (e.g., of from 80 to 800 kD, preferablyfrom 100 to 600 kD, and more preferably from 100 to 400 kD) at a highconcentration (e.g., 5-40%, preferably 10-30%, more preferably 15-25%),crosslinked with calcium. Preferably, the ratio of M units to G units inthe coat alginate is from 0.2:1 to 6:1, more preferably from 0.3:1 to3:1, and most preferably from 0.4:1 to 1.5:1.

It should be appreciated that molecular weight, concentration and M:Gratio independently affect the viscosity and permeability of thealginate. The desired viscosity and permeability are controlled bysimultaneously optimizing all three. In general, the same permeabilityis found in alginates of a lower concentration when they have a higherM:G ratio. For example, a 25% solution with M:G of 1:1 might havesimilar properties as a 12% solution with M:G of 2:1.

The overcoat alginate is selected for high permeability and highbiocompatibility, typically consisting of alginate of low concentrationcrosslinked with calcium. Preferably, the overcoat alginate permitsdiffusion of molecules and/or complexes having a molecular weight below800 kD, preferably below 400 kD, and more preferably below 200 kD.Typically, the overcoat alginate consists of alginate (e.g., of from 10to 800 kD, preferably from 20 to 400 kD, and more preferably from 20 to400 kD) at a low concentration (e.g., 0.5-10%, preferably 1-5%, morepreferably 1.5-3%), having a high proportion of M units crosslinked withcalcium. Preferably, the ratio of M units to G units in the coatalginate is from 0.2:1 to 6:1, more preferably from 0.5:1 to 2:1, andmost preferably from 0.6:1 to 1.5:1.

The core alginate is selected to provide a suitable environment for thepancreatic islets (or other cells or tissue) and for the trophic factorsand nurse cells needed. For example, given the importance of oxygendiffusion, most implants will include hemoglobin or another oxygencarrier in the alginate.

Many cells are healthier in the presence of collagen. Other trophicfactors may be suitable for particular cells and tissues. The optimalcombination of ingredients for the implant core can be evaluated usingestablished cell culture methods.

Once the components of the implant core are selected, they can becombined. Sodium (or potassium) alginate can be used with a lowconcentration of citrate to keep the mixture completely liquid. Citratestrongly chelates calcium and other multivalent cations so that theycannot crosslink and gel alginate.

FIG. 7 is a diagram of a typical mold used for the fabrication of thebioartificial implant. The molds are approximately circular, but alsomay be oval or any other compact shape. The two halves of the mold,shown separated in FIG. 7A, can be combined by touching their outerannuluses (FIG. 7B) and secured in that position with clamps (FIG. 7C)The bulk of the mold 11 is a frit made from sintered stainless steel(Mott Metalurgical, NO or GT) with a porosity between 0.01 and 5 μm,preferably approximately 0.4 μm. The frit 11 may also be made fromsintered glass or ceramic. The frit 11 is thick enough to providemechanical strength to the entire assembly FIG. 7C. The outer annulus21, 22 can be made of the same material as the frit 11 (when it isnonfragile, e.g. sintered stainless steel) or of a nonporous rigidmaterial such as stainless steel or ceramic if the frit 11 is fragile.The annulus 21 of the upper mold is flush with the frit 11. The annulus22 of the upper mold extends out from the frit 11 by a precise amountranging from 10 μm to 500 μm.

The molds may be used one to three times in the fabrication of theimplant, and may come in several dimensions suitable for each stage ofthe fabrication. A set of molds for a particular fabrication sequenceare of similar diameter, except that the inner diameter of annulus 21will be slightly larger for a mold used in a later step. The increase ininner diameter may vary from 20 μm to several hundred μm (e.g., 300-400μm), and will typically be about 50 μm. A set of molds for a particularfabrication sequence will have a successively increasing depth of thefrit from the annulus of the lower mold 22 to accommodate the formationof successive layers of the implant.

Molds optionally have a membrane 12 attached to their surfaces. Themembrane (e.g., Track-etch/Poretics, Livermore, California) can have apore size of 0.01 μm up to 0.2 μm.

The molds may be either of two types shown in FIGS. 7 and 8. The moldshown in FIG. 8 may be used to fabricate coat halves. Two premolded coathalves are required to fabricate the implant if premolded coating halvesare used. The upper mold half shown in FIG. 8 has an embossed disc sothat the mold void defines a sheet with an annular, thicker ring (calleda “coat half”).

Other fabrication steps may require molds like the ones shown in FIG. 7.The surfaces of both mold halves are preferably flat, and may have moldvoids that define perfect sheets.

Changes in the physical properties of alginate in the presence ofvarious ions are exploited in the fabrication methods. Alginate is apolymer of sugar acids. In the presence of monovalent counter ions suchas sodium and potassium, alginate solutions are liquid. In the presenceof multivalent cations such as calcium, barium and zinc, the alginatesolution forms a gel. The gelling mechanism is that the multivalentcations become ionically bonded to two acid groups in the alginatepolymer, crosslinking them. Conversely, if a chelating agent such ascitrate or EDTA is contacted with an alginate gel, the cross-linkingions become detached from the alginate gel and are sequestered by thechelating agent.

These relations are summarized in the following diagram:

gelling agent (e.g. Ca²⁺) alginate liquid alginate gel liquefying agent(e.g. citrate)

This property of alginate is exploited in three ways.

First, liquid alginate placed in a mold is gelled by allowing calcium(or other suitable multivalent cation(s)) to diffuse through the frit byimmersing the apparatus in a solution of calcium chloride (or salt(s) ofother suitable multivalent cation(s)).

Second, a liquid alginate is effectively linked to a gelled alginate bya two-step process. The first step involves contacting the gelledalginate with a chelating agent (e.g., citrate, 1-100 mM, preferably5-50 mM, more preferably approximately 10 mM) present in the liquidalginate. When the liquid alginate containing a low concentration ofchelating agent is contacted with the gelled alginate, the citrate atthe interface liquifies the surface of the alginate gel, which thenmixes with the liquid alginate.

Subsequent use of gelling agent (in a second step) then gels the liquidalginate layer and the liquified surface of the alginate gel, which has,at least in part, been mixed with the liquid alginate, thus forming astrong bond between the two layers as a result of formation of newcrosslinks between the alginate gel of the previously gelled layer(e.g., the core) and the newly-formed alginate gel of the subsequentlayer (e.g., the coat layer). In a similar fashion, alginate gel of thecoat layer can be bonded to alginate gel of the overcoat layer.

The rapidity of the crosslinking reaction can be exploited to make asmooth surface on the implant. If the membrane surface of the mold 12 iswetted with a calcium (or other multivalent cation) solution at themoment of contact with liquid alginate, the rapid gelation reactioninstantly produces a smooth surface.

The fabrication sequence begins with forming either the core or the twocoat halves, then to forming the coated core, then optionally, to addingthe overcoat.

A typical fabrication sequence is illustrated in FIGS. 4, 5 and 6. Thefrit 11 and membranes 12 of the molds are shown. Fabrication of the coreis illustrated in FIG. 4. The optional core mesh 2 is surrounded byalginate 3 and islets 1. Note that some islets touch the membrane 6. Thecore is gelled by diffusing in a crosslinking agent (e.g., calcium ion)through the frit 11.

Fabrication of the coated core is illustrated in FIG. 5. The core (1-3)is now surrounded by the coat 4. The liquid coat alginate 4, applied tothe upper and lower surfaces of the core, containing a low concentrationof citrate, mixes with the surface of the core alginate 3. Note thatwhere an islet touched the membrane of the core mold 6, it is now safelywithin the coat. The coated core is gelled by diffusing in acrosslinking agent (e.g., calcium ion) through the frit 11.

The optional overcoat 5 is added as shown in FIG. 6. The final layer,whether it be the coat or overcoat, may be prepared by wetting thesurface of an appropriate mold with a solution of crosslinking agent,and contacting the solution of crosslinking agent with the liquidalginate of the surface layer to make a smooth surface on thebioartificial implant.

Core mesh 2 may optionally be added to increase the strength of theimplant. At higher concentrations of alginate, alginate gel issufficiently strong that mesh may not be required. The mesh can be ofany monofilament or multifilament natural or synthetic polymer. Themultifilament fabric mesh shown in FIGS. 9, 10 and 11 has the furtheradvantage of allowing alginate gel to penetrate the mesh fibers,increasing bonding of the core alginate 3 to the mesh 2.

The thickness of the implant and each of its layers is controlled by themold dimensions. To produce an implant of 400 μm thickness, the coremold, for instance, may have a void depth of 300 μm. The coating moldmay then have a void depth of 350 μm, and the overcoating mold a voiddepth of 400 μm.

FIG. 3 illustrates an alternative method for fabricating the coatedcore. Here, the coat halves 4 have been fabricated in the moldillustrated in FIG. 8. Thereafter, the liquid core 1 and 3 and mesh 2are added, and the molds pressed together. Bonding between the gelledcoat halves 4 and the liquid core 3 proceeds as described above,followed by immersing the assembly in crosslinking agent (e.g., calciumion solution) to effect complete gelation of the whole coated core.

FIG. 3 also illustrates the crucial regions near the edge of the moldwhere the core and the two mold halves come together. The dimensions ofthe implant and its component layers are controlled by mold dimensionsby analogy with the description above.

An effect similar to that achieved with liquefying agent can be achievedby diffusing barium into the gel before the liquid alginate is broughtinto contact with the gelled alginate. The barium bound to the alginicacid is displaced by the monovalent cations of the liquid alginate anddiffuses out of the gel into the liquid where it interacts with residuesof the liquid alginate, thereby partially crosslinking the liquidalginate and partially liquefying the alginate gel. Subsequent exposureto chelating agent completes crosslinking of the two layers.

Another variation is to produce coating halves by schooling liquidalginate onto membranes, which can be peeled away from the newlyfabricated coated core by wetting with liquefying agent.

Numerous other variations in the sequence of gelation, crosslinking anddiffusion of gelling and liquefying agents exist, some of which aredescribed in the Examples.

The present invention is further illustrated by the following specificbut non-limiting examples. Percents are given in weight percents andtemperature in degrees Centigrade unless otherwise specified. Allsolutions are aqueous unless otherwise indicated.

EXAMPLE 1 Na Alginate Solution Preparation

Briefly, a solution of Na alginate (LV Alginate/Kelco Division of Merck& Co.) was clarified by filtration and treated with activated charcoal(perchlorate bleached). The resulting solution was precipitated byadjusting the pH to 2 with HCl. The precipitate was redissolved in 120mM NaCl-5 μM EDTA-10 mM HEPES solution and reprecipitated by addition ofethanol. The precipitate was partially redissolved in 1 M KCl, and theremaining insoluble fraction was dissolved in 120 mM NaCl-5 μM EDTA-10μM HEPES solution and reprecipitated by addition of ethanol. The finalprecipitate was washed thoroughly with ethanol and dried in vacuo at 80°C.

To prepare alginate solutions of under 5% concentration, the resultingdry material was redissolved in 10 mM HEPES-10 mM Na citrate-110 mMNaCl, dialyzed (10 kD) against 10 mM HEPES-10 mM Na citrate-110 mM NaCl,and filtered through a 0.1 μm membrane.

To prepare alginate solutions of over 5% concentration, the resultingdry material was redissolved to a 1% solution, dialyzed (10 kD) againstH₂O, then filtered through a 0.1 μm membrane. The alginate was sterilelylyophilized then redissolved to the desired concentration in sterile 10mM HEPES-10 mM Na citrate-110 mM NaCl.

EXAMPLE 2 Islet Suspension Preparation

The preparation was performed under sterile conditions.

Islets isolated from rats were washed with isotonic NaCl and suspendedin a 2% alginate solution (prepared by the procedure of Example 1) at aconcentration of 150,000 islets per milliliter.

EXAMPLE 3 Bioartificial Implant Preparation by Molded Gum Coat Method

The preparation was performed under sterile conditions.

Using the coating mold, the two halves of the coat were prepared asfollows. A thin plastic film was applied to the upper coating mold. A20% Na alginate solution was prepared according to the method in Example1, then a quantity sufficient to produce a 20 μm alginate half coat wasapplied to the lower mold. This material is considered a gum because,although liquid, it is viscous due to the high concentration ofalginate.

The two mold halves were pressed together. The upper mold was removed,and the thin plastic film carefully peeled off the coat half. The secondcoat half was prepared in the same way.

One half of the islet suspension described in Example 2 was placed onthe prepared coat half remaining in the lower coating mold. A mesh(Allied Silicone, Ventura, California) was cut to a size slightlysmaller than the inner diameter of the mold (so as to fit within thedepression in the prepared coat half reserved for the core). Theremaining one half of the islet suspension was placed on the mesh in thelower coating mold. The total volume of mesh and suspension was chosento exactly fill the void between the coat halves. The other lower coatmold was inverted and carefully pressed on to the first coating mold andclamped. Because both the core and coat alginates are liquid, theydiffused into each other immediately. The assembly was immersed in a 120mM CaCl₂-10 mM HEPES solution for 30 minutes to crosslink the alginate.The two mold halves were separated and the coated core removed.

The upper and lower overcoat molds were wetted with a solution of 120 mMCaCl₂-10 mM HEPES. A 2% solution of Na alginate was prepared by themethod of Example 1 in a volume sufficient to form a 20 μm overcoat. Onehalf of the Na alginate solution was placed in the overcoat mold. Thecoated core was washed with aqueous 120 mM NaCl-10 mM HEPESexhaustively, and was carefully placed in the overcoat mold. The secondhalf of the Na alginate solution was placed in the overcoat mold. Theupper overcoat mold was pressed onto the lower overcoat mold andclamped. The assembly was immersed in an aqueous 120 mM CaCl₂-10 mMHEPES solution for 30 minutes to crosslink the alginate. The two moldhalves were separated and the overcoated coated core (completebioartificial implant) removed.

EXAMPLE 4 Bioartificial Implant Preparation by Partial CoatLiquification

The preparation was performed under sterile conditions.

Using the coating mold, the two halves of the coat were prepared asfollows. A 20% Na alginate solution was prepared according to the methodin Example 1, then a quantity sufficient to produce a 20 μm alginatehalf coat was applied to each of the lower coat molds. The two uppermolds were placed on the two lower molds, pressed together and clamped.The assembly was immersed in a 120 mM CaCl₂-10 mM HEPES solution for 30minutes to crosslink the alginate.

The upper mold was removed and the coats washed exhaustively in placewith 120 mM NaCl-10 mM HEPES. One half of the islet suspension describedin Example 2 was placed on the prepared coat half remaining in the lowercoating mold. A mesh (Allied Silicone, Ventura, California) was cut to asize slightly smaller than the inner diameter of the mold (so as to fitwithin the depression in the prepared coat half reserved for the core).The remaining one half of the islet suspension was placed on the mesh inthe lower coating mold. The total volume of mesh and suspension waschosen to exactly fill the void between the coat halves. The other lowercoat mold was inverted and carefully pressed onto the first coating moldand clamped. After 5 minutes incubation to allow partial dissolution ofthe coat alginate gel by interaction with citrate in the islet alginatesuspension, the assembly was immersed in a 120 mM CaCl₂-10 mM HEPESsolution for 30 minutes to crosslink the alginate core and couple it tothe coat. The two mold halves were separated and the coated coreremoved.

The upper and lower overcoat molds were wetted with a solution of 120 mMCaCl₂-10 mM HEPES. A 2% solution of Na alginate was prepared by themethod of Example 1 with a volume sufficient to form a 20 μm overcoat.One half of the Na alginate solution was placed in the overcoat mold.The coated core was washed with 120 mM NaCl-10 mM HEPES exhaustively,and was carefully placed in the overcoat mold. The second half of the Naalginate solution was placed in the overcoat mold. The upper overcoatmold was pressed on to the lower overcoat mold and clamped. The assemblywas immersed in a 120 mM CaCl₂-10 mM HEPES solution for 30 minutes tocrosslink the alginate. The two mold halves were separated and theovercoated coated core (complete bioartificial implant) removed.

EXAMPLE 5 Bioartificial Implant Preparation by Partial Core-CoatInterface Liquification

The preparation was performed under sterile conditions.

Using the coating mold, the two halves of the coat were prepared asfollows. A 20% Na alginate solution was prepared according to the methodin Example 1, then a quantity sufficient to produce a 20 μm alginatehalf coat was applied to each of the lower coat molds. The two uppermolds were placed on the two lower molds, pressed together and clamped.The assembly was immersed in a 120 mM CaCl₂-10 mM HEPES solution for 30minutes to crosslink the alginate.

The upper mold was removed and the coats washed exhaustively in placewith 120 mM NaCl-10 mM HEPES. One half of the islet suspension describedin Example 2 was placed in the lower core mold. A mesh (Allied Silicone,Ventura, California) was cut to slightly smaller than the inner diameterof the mold (so as to fit within the depression in the mold reserved forthe core). The remaining one half of the islet suspension was placed onthe mesh in the lower core mold. The total volume of mesh and suspensionwas chosen to exactly fill the void between the coat halves. The twomolds were pressed together and clamped. The assembly was immersed in a120 mM CaCl₂-10 mM HEPES solution for 30 minutes to crosslink thealginate.

The upper and lower core mold were separated and the core removed. A fewdrops of 1.5% Na alginate-10 mM Na citrate-10 mM HEPES-110 mM NaCl wereapplied to the center of one of the coating halves in its coating moldhalf. The core was placed on top and a few drops of 1.5% Na alginate-10mM Na citrate-10 mM HEPES-110 mM NaCl were applied to the top of thecore. The other coat half in its coating mold half was pressed down ontop and the entire assembly clamped. The assembly was immersed in a 120mM CaCl₂-10 mM HEPES solution for 30 minutes to couple the alginate coreto the coat. The two mold halves were separated and the coated coreremoved.

The upper and lower overcoat molds were wetted with a solution of 120 mMCaCl₂ -10 mM. HEPES. A 2% solution of Na alginate was prepared by themethod of Example 1 with a volume sufficient to form a 20 μm overcoat.One half of the Na alginate solution was placed in the overcoat mold.The coated core was washed with 120 mM NaCl-10 mM HEPES exhaustively,and was carefully placed in the overcoat mold. The second half of the Naalginate solution was placed in the overcoat mold. The upper overcoatmold was pressed on to the lower overcoat mold and clamped. The assemblywas immersed in a 120 mM CaCl₂-10 mM HEPES solution for 30 minutes tocrosslink the alginate. The two mold halves were separated and theovercoated coated core (complete bioartificial implant) removed.

EXAMPLE 6 Bioartificial Implant Preparation by Diffusion Coat Method

The preparation was performed under sterile conditions.

One half of the islet suspension described in Example 2 was placed inthe lower core mold. A mesh (Allied Silicone, Ventura, California) wascut to slightly smaller than the inner diameter of the mold (so as tofit within the depression in the mold reserved for the core). Theremaining one half of the islet suspension was placed on the mesh in thelower core mold. The total volume of mesh and suspension was chosen toexactly fill the void between the coat halves. The two molds werepressed together and clamped. The assembly was immersed in a 120 mMCaCl₂-10 mM HEPES solution for 30 minutes to crosslink the alginate.

The upper and lower core mold were separated and the core removed andimmersed in 120 mM BaCl₂-10 mM HEPES for 5 minutes to effect an exchangereaction between Ca and Ba ions. The core was then washed exhaustivelywith 120 mM NaCl.

A solution of 20% Na alginate prepared by the method of Example 1 ofsufficient volume to product a 20 μm coat was prepared and half of thevolume applied to the lower coat mold. The core was placed on top,overlaid with the remaining alginate solution, and incubated for 5minutes to allow sodium in the coat alginate solution to exchange withbarium bound to the core alginate and for liberated barium to diffuseinto the coat alginate and crosslink it. The assembly was then immersedin a 120 mM CaCl₂-10 mM HEPES solution for 30 minutes to crosslink thealginate. The two mold halves were separated and the coated coreremoved.

The upper and lower overcoat molds were wetted with a solution of 120 mMCaCl₂-10 mM HEPES. A 2% solution of Na alginate was prepared by themethod of Example 1 with a volume sufficient to form a 20 μm overcoat.One half of the Na alginate solution was placed in the overcoat mold.The coated core was washed with 120 mM NaCl-10 HEPES exhaustively, andwas carefully placed in the overcoat mold. The second half of the Naalginate solution was placed in the overcoat mold. The upper overcoatmold was pressed on to the lower overcoat mold and clamped. The assemblywas immersed in a 120 mM CaCl₂-10 mM HEPES solution for 30 minutes tocrosslink the alginate. The two mold halves were separated and theovercoated coated core (complete bioartificial implant) removed.

EXAMPLE 7 Pancreatic Islet Implant into Diabetic Mice (IP)

Host Balb/C mice were rendered diabetic by IP injection of 250 mg/kg ofstreptozotocin at 50 mg/ml in 0.1 M citrate buffer, pH=4.5, several daysprior to implantation. Bioartificial implants prepared according toExample 4 containing 2000-3000 islets were inserted into the peritonealcavity through an abdominal incision and the mice were sutured.

What is claimed as new and is desired to be secured by Letters Patent ofthe United States is:
 1. A process for making a biocompatible implant,comprising the steps of: combining living tissue or cells to beimplanted with a solution of an alginate, the concentration of alginatebeing sufficient to form a gel in the presence of a crosslinking agent;contacting the resulting mixture with a solution of crosslinking agentin a concentration sufficient to gel said alginate; and optionally,forming a coat layer thereon, said coat layer comprising a biocompatablealginate gel.
 2. The process of claim 1, wherein said coat layer isformed by contacting said gelled alginate with a liquid alginatecontaining sufficient chelating agent to liquify at least part of saidgel.
 3. A method of making a physiologically active and biocompatiblecellular implant for implantation into a host body, said methodcomprising steps of: (a) forming first and second films of first andsecond substantially uncross-linked polymer solutions, (b) forming asandwich of a cell suspension layer of physiologically active cells in asubstantially uncross-linked third polymer solution between said firstand second films, and (c) diffusing cross-linking agent through saidfirst and second films to the cell suspension layer until the first andsecond polymer solutions of said first and second films, respectively,and said cell suspension layer are cross-linked to form a gelledcellular implant.